16α-Hydroxy Steroids: XI. 2β- and 16α-Hydroxylation of 9α-Fluorohydrocortisone by Strains of Streptomyces roseochromogenes

9α-Fluorohydrocortisone is hydroxylated by Streptomyces roseochromogenes in the 16α-, the 2β-, and in both the 16α- and 2β- positions. 16α-Hydroxylation appears to be the more predominant and rapid pathway of transformation. The degree of 2β-hydroxylation is dependent upon the strain used and may be increased to the point of its being a major route or virtually eliminated by the proper selection of isolates.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The -isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and -ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate -ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the -ketoisocaproate-mediated inhibition is discussed.Abbreviations IPM -isopropylmalate - KIC -ketoisocaproate - KIV -ketoisovalerate - DTNB 5:5 Dithiobis-(2-nitrobenzoe acid)     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

-Isopropylmalate (IPM) synthase, the first enzyme in the biosynthesis of l-leucine, was purified to a specific activity of 12 mole/min x mg protein from the valine-isoleucine double auxotrophic mutant A-81 of the hydrogen bacterium Alcaligenes eutrophus H 16. The activity in crude extracts of derepressed cells was 0.106 moles of isopropylmalate formed per min and per mg protein. Gel electrophoresis and regel electrophoresis of the isolated main band resulted in several distinct bands, which were not altered by the additions of substrate -ketoisovalerate, feedback inhibitor leucine or other effectors.The isoelectric points of the enzyme protein was between 3.9 and 4.0. The molecular weight was 114500 daltons and 100000 respectively in the absence and presence of the feedback inhibitor leucine. The enzyme activity depended strongly on the pH, the optimum is at pH 8.2. The enzyme was could labile and exhibits temperature anomalies.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

LRP16与 ERα相互作用增强ERα介导的转录活性

LRP16是一个雌激素（E2）通过受体α（ERα）诱导表达的靶基因,LRP16不仅促进人乳腺癌MCF-7细胞的增殖,而且促进细胞的侵袭生长,但对LRP16作用的分子机制尚不清楚.首先,检测到LRP16表达缺陷明显削弱了MCF-7细胞对E2的反应性增殖能力;采用Northern 印迹与Western印迹法,进一步检测到抑制LRP16表达,明显损害了ERα靶基因对E2诱导上调的反应性,这些基因包括了cyclin D1, c-myc, c-fos,MTA3, pS2和维甲酸受体α基因（RARα）等;以上结果提示,LRP16参与了ERα介导的信号途径.将ERα模式启动子报告基因3×ERE-TATA-luc,ERα以及LRP16表达载体共转染MCF-7与HeLa细胞.结果发现,LRP16增强了ERα对报告基因的转录激活,并呈现对LRP16的剂量依赖性;进而采用GST-pull down以及免疫共沉淀方法证实了LRP16与ERα之间的直接相互作用,该作用不依赖于E2的存在,但可被E2增强;采用哺乳动物双杂交方法进一步证实了ERα与LRP16相互作用位点存在于A/B区的激活功能域-１（AF1）.以上结果表明,LRP16是ERα的一个共激活因子,通过相互作用,LRP16增强了ERα介导转录活性.该研究为LRP16促进ERα阳性乳腺癌细胞增殖与侵袭生长提供了合理的分子解释.     

微生物合成11α-羟基-16α,17α-环氧孕酮的动力学

对耐温黑根霉菌丝体催化16α,17-α-氧孕酮生成11α-羟基-16α,17α-环氧孕酮的反应体系进行了研究,考察了不同反应时间、不同菌体量和底物质量浓度对11α-羟化反应的影响,建立了相应的动力学模型,其方程为r=0.031Sr/1.05＋Sr。结果表明：提高菌体质量浓度有利于提高反应速率;底物浓度与反应初速率的关系与米氏方程相似。     

Species and Tissue Distribution of Proteins Binding 16α,17α-Cycloalkanoprogesterone Derivatives

The binding of [³H]progesterone and [³H]16,17-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16,17-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [³H]16,17-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16,17-cycloalkanoprogesterones and exhibits submicromolar K _d values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.     

Unusual oxidative transformations of a steroidal 16α,17α,22-triol

A number of unexpected reactions were observed during attempts to invert configuration at C16 in 16α,17α,22-triol 3a. The PDC oxidation of 3a produced the D-seco-aldehyde 4a. Analogous compound 4b was obtained by Swern oxidation of the 16α,17α-dihydroxy-22-O-TES-ether 3b in addition to the desired 16-ketone 7. The unprotected triol 3a yielded pentacyclic products 5 and 6 under similar conditions. The Mitsunobu reaction of the triol 3a afforded 16-ketone 8 with inverted configuration of the side chain. During heating of a solution of 3a in THF with NaH at reflux autoxidation to the 16-ketone cyclic hemiketal 5, identical to one of the Swern oxidation products, took place.     

16α-hydroxylation of progesterone by a strain ofactinomyces globosus

В систематическое исследование трансформации свойств Actinomycetes было было установлено, что 17 из 76 видов тестировани е преобразованы прогестерон по 16 га-гидрокси -п рогестерона. Оптимальные условия для этой трансформац ии были изучены этой трансформации б ыли изучены следующие результат ы:

1. (1)
   Оптимальное рН для данного типа трансформации была 6–7. На нижней hydroxylation ценностей была меш ает.     
   
   

大鼠TGF－α修饰16肽的溶液构象

已知大鼠Ｔ０Ｆ－α的抗原部位干Ｃ环,且大鼠的ＴＧＦ—α（３４－４３）和ＴＧＦ－α（４４－５０）均有较强的活性以转化正常细胞。为了揭示其结构与功能关系,合成了大鼠ＴＧＦ－α修饰１６肽。在前文用二维核磁共振技术归属质子谱并验证其一级结构的基础上,本文测定了不同混合时间的二维ＮＯＥＳＹ谱。根据所得的数据因原始斜率法求出距离约束。通过约束分子动力学计算定出该分子的溶液构象。其中还用ＤＱＦ－ＣＯＳＹ的数据求出二面角作为决定溶液构象的参考。     

16α,17α-Cycloalkane Derivatives of Progesterone Intensively Bind to a Rat Serum Protein

The interaction of 6-methyl-[1,2-³H]16,17-cyclohexanoprogesterone with rat serum proteins has been studied. Specific binding of this ligand characterized by K _d = 0.36 ± 0.10 M and concentration of binding sites (B_max) of about 1 M (27.8 ± 12.5 pmol/mg total protein) was found. According to competitive analysis, the affinity of the studied progestins to a protein that differs from transcortin was to some extent correlated with their hydrophobicity. The dissociation kinetics of ³H-ligand–protein complexes were biphasic, the binding sites forming stable and labile complexes with ³H-ligand being eluted in the same region during ion-exchange chromatography. In overall properties, the serum protein differs from the progesterone receptor and the pregna-D"-pentarane-specific protein from rat uterus. It is suggested that the revealed protein may provide high progestagenic activity of 6-methyl-16,17-cyclohexanoprogesterone by prolonging its retention in the bloodstream.     

Immunological determination of hydroxylated 6α-fluor-16α-methyl deoxycorticosterone derivatives in fungal steroid transformation

Summary Antisera and monoclonal antibodies were raised against the 9 and 11 hydroxylated derivatives of 6-fluor-16-methyl deoxycorticosterone via 3-0-carboxy-methyloxim-bovine serum albumin immunogenic conjugates. The properties of the antibodies and several immunoassay systems were evaluated including an enzyme immunoassay with nitrocellulose as the solid phase and an insoluble, colored reaction product allowing visual evaluation of assays. The performance of the techniques was evaluated for the industrial important fungus, Curvularia lunata.Abbreviations AP alkaline phosphatase - BCIP 5-bromo-4-chloro-3-indolyl phosphate - BSA bovine serum albumin - DMF dimethyl formamide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - ELISA enzyme linked immunosorbent assay - FMDOC 6-fluor-16-methyl deoxycorticosterone - MAB monoclonal antibodies - NC nitrocellulose - RIA radioimmunoassay - TBS TRIS buffered saline - TLC thin layer chromatography Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

Aryl hydrocarbon hydroxylase and 16α-hydroxylase in cultured human lymphocytes

Aryl hydrocarbon hydroxylase and 16-hydroxylase were examined in intact, cultured human lymphocytes. The two microsomal mixed-function oxygenases had different pH optima and showed competitive inhibition for enzyme induction and activity. Population distributions were lognormal for both enzymes, giving apparent evidence for polygenic control. Induced levels of AHH were slightly higher among first-order relatives of lung or colon cancer patients than in the control group. The correlation coefficient (r) for AHH and SAH co-inducibility was –0.08, indicating no correlation and suggesting the absence of association between the two enzymes in man.Supported in part by NIH Contract NO1-CP5-5626 and NTSU Faculty Grants 34984 and 34815.National Science Foundation Predoctoral Trainee.     

5-α-androst-16en-3α-on: A male pheromone? A brief report

Olfactory cues play a prominent, yet underestimated role in shaping emotional attitudes towards conspecifics. Among humans olfactory cues can have effects on behavior. In a rating study (n = 290) females rated the smell of androstenone. The emotional reaction to androstenone changed during the menstrual cycle. Females rated the main component of male body odor unattractive. This changed to a neutral emotional response at the conceptive optimum around ovulation. The finding has direct consequences for hypotheses concerning the evolutionary loss of estrus. It is suggested that the cyclic-dependent emotional rating of androstenone may facilitate active female choice of sex partners and may be a proximate cue for female mate-choice.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

A Method for Preparing 16α-Hy-droxypregnenolone-3-succinate by Streptomyces roseochromogenes

A choline dehydrogenase, which was present in the particulate fraction of the cell-free extract of Pseudomonas aeruginosa A-16, oxidized choline to betaine aldehyde without any dissociable coenzymes, while the enzyme, which was treated with Triton X-100, oxidized choline only with a supplement of phenazine methosulfate. The difference spectrum showed the presence of cytochrome-like components in the particulate. Km values for choline and phenazine methosulfate were 1.7 × 10^?3 m and 1.4 × 10^?4 m, respectively. The dehydrogenase was inhibited by SH-reagents such as p-chloromercuribenzoate and iodoacetic acid. Of a variety of substrates tested, only choline caused the enzymatic reduction of phenazine methosulfate. The estimation of choline was tried using the enzyme.     

An assay for urinary estriol-16α-glucuronide based on antibody-enhanced chemiluminescence

An immunoassay for estriol-16α-glucuronide in pregnancy urine is described that utilizes antibody-enhanced chemiluminescence. The steroid glucuronide was covalently conjugated with the chemiluminescent marker aminobutyl-ethyl-isoluminol. The light yield of this conjugate upon oxidation was augmented by specific antibody, and this effect was inhibited by addition of the homologous steroid glucuronide (10–100 pg) in a dose-dependent manner. The assay does not require separation of bound and free ligand and proved satisfactory with respect to sensitivity, precision and accuracy. Assay results obtained by radioinununoassay and chemiluminescence immunoassay were in good agreement (r = 0.98; n = 25).     

生物转化法合成16α-羟基泼尼松龙的发酵工艺研究

目的:研究1株玫瑰产色链霉菌(Streptomyces roseochromogenes)的发酵培养基和底物转化条件,以提高16α-羟基泼尼松龙的转化率。方法:采用紫外与氯化锂复合诱变获得目的菌株TS-58,利用正交实验等方法考查摇瓶发酵条件,研究不同浓度的碳源、氮源对玫瑰产色链霉菌生长的影响,以及不同底物浓度、底物加入时间、装液量、金属离子和添加助溶剂等条件对转化生成16α-羟基泼尼松龙能力的影响。结果:获得最佳转化培养基为葡萄糖10 g/L、可溶性淀粉50 g/L、蛋白胨10 g/L、黄豆饼粉25 g/L、磷酸二氢钾0.2 g/L、硫酸镁0.5 g/L硫酸锌0.5 g/L。在底物投料量5 mg/mL添加0.8 mg/ml PEG助溶剂的最优条件下,16α-羟基泼尼松龙的转化率达到了13.8%。结论:突变株Streptomyces roseochromogenes TS-58能有效地在泼尼松龙上引入16α-羟基羟基,为工业生产16α-羟基泼尼松龙奠定了基础。